The present invention relates to methods for evaluating aggregated forms of amyloid polypeptides. The invention further relates to methods for testing potential inhibitors of amyloidosis in vitro or in vivo in order to develop new therapies for diseases or disorders associated with amyloidosis, such as Alzheimer""s disease.
Alzheimer""s disease is characterized by the presence of cerebral amyloid plaques deposited around nerve cells, which eventually erode and destroy the normal processes of the brain. The primary protein component of such plaques is the aggregated form of the 39 to 43 residue isoforms of the xcex2-amyloid protein (Axcex2). The primary pathogenic event in Alzheimer""s disease is the formation of these fibrillar amyloid plaques in the brain parenchyma and vasculature. Deposition of fibrillar aggregates of these amyloid polypeptides has been shown to be due to the self assembly of the polypeptides into macromolecular structures consisting of a cross-xcex2-sheet conformation, which is toxic to cultured neuronal cells.
A goal of treating Alzheimer""s disease is to prevent aggregate formation and/or to therapeutically administer an inhibitor of aggregate formation after the disease has been diagnosed, in order to reverse the toxic effects of the protein. Agents that block the formation of macromolecular aggregates can thus potentially act to inhibit cytotoxicity and are of pharmaceutical interest because they can potentially act as anti-Alzheimer""s drugs. Among the several small molecules reported to be aggregation inhibitors, Congo red, a biphenyl diazo sulphonated dye, is reported to be a neuroprotective compound (Pollack et al., Neurosci. Lett., 1995, 197:211-214; Lorenzo et al, Proc. Natl. Acad. Sci. USA, 1994, 91:12243-12247; and Burgevin et al., NeuroReport, 1994, 5:2429-2432).
PCT Application PCT/US93/06637 (the xe2x80x9c""637 applicationxe2x80x9d) describes the use of Congo red and its pharmaceutically acceptable salts, or derivatives thereof, for treating amyloidogenic diseases, among them Alzheimer""s disease. Congo red reportedly interferes with amyloidosis or destabilizes already formed amyloid structures. These properties establish a basis for the ""637 application to advocate administration of Congo red for treatment of conditions associated with the deposition of amyloidogenic protein in plaques. Congo red, as a therapeutic agent, may be administered in vivo in sufficient quantity to interfere with amyloidogenic protein formation or the destabilization of amyloid already formed. Thus, Congo red reportedly not only binds amyloid plaques, but can also inhibit the accumulation of amyloidogenic protein.
Congo red""s value and effectiveness as a possible treatment for Alzheimer""s disease require further examination because Congo red cannot cross the blood brain barrier. Derivatives, analogues, salts, etc. of Congo red may have the same mode of action on xcex2-amyloid peptides as Congo red, i.e., such derivatives may also be neuro-protective.
Currently, antibody-based methods and fluorescence methods using Thioflavin T are being used to detect aggregated peptide for screening anti-Alzheimer""s agents. A fluorescence correlation spectroscopy method has been used to determine polymerization of amyloid peptide (Tjernberg et al., Chemistry and Biology, 1999, 6:53-62). However, these techniques do not differentiate various conformational forms of aggregated polypeptide.
An assay for detecting alternate conformational forms of aggregated amyloid polypeptide is not disclosed in the prior art. Such assays optimally can be used to rapidly assess the aggregation state of the polypeptide, especially in the presence of inhibitors of fibrillogenesis.
Thus, there is a need in the art for methods for probing amyloidosis and amyloid plaques, which in turn addresses the need to study the process by which amyloidogenic proteins cause Alzheimer""s disease. Specifically, there is a need to develop assays involving biophysical and analytical methods to characterize the conformation and aggregation state of aggregates of amyloid polypeptide and to discriminate the effect of potential inhibitors of amyloidosis, specifically in Alzheimer""s disease. Since very little is known about the nature of toxic aggregates in Alzheimer""s disease and the mechanism of formation of these aggregates in the brain, a combination of assays are necessary to determine if potential inhibitors are producing the desired effect in in vitro test systems. Assays are also needed to determine the effect of test compounds on conformation of amyloid polypeptide in bodily fluids such as plasma or cerebral spinal fluid.
This invention provides methods to detect various aggregated forms of amyloid polypeptide, for example, using amyloid-specific dyes, such as Congo red and other related analogs, or fluorophore-labeled amyloid peptides. The methods are useful in determining the aggregated state of the polypeptide as well as to test modulators of aggregation in in vitro models. The method can be used in determining the stage at which intervention in the process of aggregation reduces toxicity, e.g., Axcex2 peptide to neuronal cells. The methods can also be used to determine the effects of inhibitor or anti-Alzheimer""s agent for therapeutic or prophylactic treatment of amyloidosis or other related diseases.
Thus, in one aspect, the present invention provides a method for determining an aggregation conformation of an amyloid polypeptide. This method comprises correlating a spectroscopic property of a complex of an amyloid-specific spectroscopic probe, such as a sulphonated diazo dye or fluorophore-labeled amyloid peptide contacted with the amyloid polypeptide under conditions that permit aggregation of the polypeptide, with a predetermined spectroscopic property of a complex of the amyloid-specific probe with aggregated amyloid polypeptide of known conformation.
In a further aspect, the invention provides a method for detecting an effect of a test compound for inhibition, disruption, or disaggregation of amyloid formation. This method comprises correlating spectroscopic properties of (a) a complex of an amyloid-specific spectroscopic probe, such as a sulphonated diazo dye or a fluorophore-labeled amyloid peptide, contacted with an amyloid polypeptide and a test compound under conditions that permit aggregation of the polypeptide, and (b) a complex of the amyloid-specific spectroscopic probe contacted with the amyloid polypeptide under conditions that permit aggregation (control). The spectroscopic probe can be contacted with the sample prior to aggregation or, if it is an amyloid-specific dye, after aggregation. If the spectroscopic probe is a fluorophore-labeled peptide, it should be permitted to aggregate with the amyloid polypeptide. The test compound can be added prior to or after aggregation. A difference in the spectroscopic properties indicates that the test compound has an effect on amyloid formation.
These and other aspects of the invention will be better understood by reference to the Drawings, Detailed Description, and the Examples.